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Enhancer binding proteins (EBPs) are key players of σ54‐regulation that control transcription in response to environmental signals. In the anaerobic microorganism Desulfovibrio vulgaris Hildenborough (DvH), orp operons have been previously shown to be coregulated by σ54‐RNA polymerase, the integration host factor IHF and a cognate EBP, OrpR. In this study, ChIP‐seq experiments indicated that the OrpR regulon consists of only the two divergent orp operons. In vivo data revealed that (i) OrpR is absolutely required for orp operons transcription, (ii) under anaerobic conditions, OrpR binds on the two dedicated DNA binding sites and leads to high expression levels of the orp operons, (iii) increasing the redox potential of the medium leads to a drastic down‐regulation of the orp operons expression. Moreover, combining functional and biophysical studies on the anaerobically purified OrpR leads us to propose that OrpR senses redox potential variations via a redox‐sensitive [4Fe–4S]2+ cluster in the sensory PAS domain. Overall, the study herein presents the first characterization of a new Fe–S redox regulator belonging to the σ54‐dependent transcriptional regulator family probably advantageously selected by cells adapted to the anaerobic lifestyle to monitor redox stress conditions. [ABSTRACT FROM AUTHOR]

Abstract Background The σ54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ54 remain unknown. Results For systematic analysis of the regulatory functions of σ54, we performed comparative genomic reconstruction of transcriptional regulons of σ54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ54 regulons between different Clostridiales species. It is proposed that σ54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species. Conclusions This study reveals previously unrecognized functions of σ54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species.

The AdhR regulatory protein is an activator of σ54-dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii. Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA+ domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ54 showed large shifts in intracellular redox state indicated by the NADH/NAD+ ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe2+-activated transcriptional regulator AdhR and σ54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckii. [ABSTRACT FROM AUTHOR]

Bacterial biofilm formation during chronic infections confers increased fitness, antibiotic tolerance, and cytotoxicity. In many pathogens, the transition from a planktonic lifestyle to collaborative, sessile biofilms represents a regulated process orchestrated by the intracellular second-messenger c-di-GMP. A main effector for c-di-GMP signaling in the opportunistic pathogen Pseudomonas aeruginosa is the transcription regulator FleQ. FleQ is a bacterial enhancer-binding protein (bEBP) with a central AAA+ ATPase σ54-interaction domain, flanked by a C-terminal helix-turn-helix DNA-binding motif and a divergent N-terminal receiver domain. Together with a second ATPase, FleN, FleQ regulates the expression of flagellar and exopolysaccharide biosynthesis genes in response to cellular c-di-GMP. Here we report structural and functional data that reveal an unexpected mode of c-di-GMP recognition that is associated with major conformational rearrangements in FleQ. Crystal structures of FleQ's AAA+ ATPase domain in its apo-state or bound to ADP or ATP-γ-S show conformations reminiscent of the activated ring-shaped assemblies of other bEBPs. As revealed by the structure of c-di-GMP--complexed FleQ, the second messenger interacts with the AAA+ ATPase domain at a site distinct from the ATP binding pocket. c-di-GMP interaction leads to active site obstruction, hexameric ring destabilization, and discrete quaternary structure transitions. Solution and cell-based studies confirm coupling of the ATPase active site and c-di-GMP binding, as well as the functional significance of crystallographic interprotomer interfaces. Taken together, our data offer unprecedented insight into conserved regulatory mechanisms of gene expression under direct c-di-GMP control via FleQ and FleQ-like bEBPs. [ABSTRACT FROM AUTHOR]

Abstract: Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA+ protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA+ domain of one well-studied bEBP in complex with its substrate, the σ54 subunit of RNA polymerase. Our results demonstrate that the free AAA+ domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ54 binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs. [Copyright &y& Elsevier]

Abstract: Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA+ (ATPases Associated with various cellular Activities) protein family. They are responsible for σ54-dependent transcription activation during infection and function under many stressful growth conditions. The majority of bEBPs are regulated in their formation of ring-shaped hexameric self-assemblies via an amino-terminal domain through its phosphorylation or ligand binding. In contrast, the Escherichia coli phage shock protein F (PspF) is negatively regulated in trans by phage shock protein A (PspA). Up to six PspA subunits suppress PspF hexamer action. Here, we present biochemical evidence that PspA engages across the side of a PspF hexameric ring. We identify three key binding determinants located in a surface-exposed ‘W56 loop’ of PspF, which form a tightly packed hydrophobic cluster, the ‘YLW’ patch. We demonstrate the profound impact of the PspF W56 loop residues on ATP hydrolysis, the σ54 binding loop 1, and the self-association interface. We infer from single-chain studies that for complete PspF inhibition to occur, more than three PspA subunits need to bind a PspF hexamer with at least two binding to adjacent PspF subunits. By structural modelling, we propose that PspA binds to PspF via its first two helical domains. After PspF binding-induced conformational changes, PspA may then share structural similarities with a bEBP regulatory domain. [Copyright &y& Elsevier]

Abstract: AAA proteins (ATPases Associated with various cellular Activities) are involved in almost all essential cellular processes ranging from DNA replication, transcription regulation to protein degradation. One class of AAA proteins has evolved to adapt to the specific task of coupling ATPase activity to activating transcription. These upstream promoter DNA bound AAA activator proteins contact their target substrate, the σ54-RNA polymerase holoenzyme, through DNA looping, reminiscent of the eukaryotic enhance binding proteins. These specialised macromolecular machines remodel their substrates through ATP hydrolysis that ultimately leads to transcriptional activation. We will discuss how AAA proteins are specialised for this specific task. This article is part of a Special Issue entitled: AAA ATPases: structure and function. [Copyright &y& Elsevier]

Abstract Background Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. Results We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. Conclusion Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm.

Enhancer binding proteins (EBPs) are key players of σ 54 -regulation that control transcription in response to environmental signals. In the anaerobic microorganism Desulfovibrio vulgaris Hildenborough (DvH), orp operons have been previously shown to be coregulated by σ 54 -RNA polymerase, the integration host factor IHF and a cognate EBP, OrpR. In this study, ChIP-seq experiments indicated that the OrpR regulon consists of only the two divergent orp operons. In vivo data revealed that (i) OrpR is absolutely required for orp operons transcription, (ii) under anaerobic conditions, OrpR binds on the two dedicated DNA binding sites and leads to high expression levels of the orp operons, (iii) increasing the redox potential of the medium leads to a drastic down-regulation of the orp operons expression. Moreover, combining functional and biophysical studies on the anaerobically purified OrpR leads us to propose that OrpR senses redox potential variations via a redox-sensitive [4Fe-4S] 2+ cluster in the sensory PAS domain. Overall, the study herein presents the first characterization of a new Fe-S redox regulator belonging to the σ 54 -dependent transcriptional regulator family probably advantageously selected by cells adapted to the anaerobic lifestyle to monitor redox stress conditions., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

The AdhR regulatory protein is an activator of σ 54 -dependent transcription of adhA1 and adhA2 genes, which are required for alcohol synthesis in Clostridium beijerinckii Here, we identified the signal perceived by AdhR and determined the regulatory mechanism of AdhR activity. By assaying the activity of AdhR in N-terminally truncated forms, a negative control mechanism of AdhR activity was identified in which the central AAA + domain is subject to repression by the N-terminal GAF and PAS domains. Binding of Fe 2+ to the GAF domain was found to relieve intramolecular repression and stimulate the ATPase activity of AdhR, allowing the AdhR to activate transcription. This control mechanism enables AdhR to regulate transcription of adhA1 and adhA2 in response to cellular redox status. The mutants deficient in AdhR or σ 54 showed large shifts in intracellular redox state indicated by the NADH/NAD + ratio under conditions of increased electron availability or oxidative stress. We demonstrated that the Fe 2+ -activated transcriptional regulator AdhR and σ 54 control alcohol synthesis to maintain redox homeostasis in clostridial cells. Expression of N-terminally truncated forms of AdhR resulted in improved solvent production by C. beijerinckii IMPORTANCE Solventogenic clostridia are anaerobic bacteria that can produce butanol, ethanol, and acetone, which can be used as biofuels or building block chemicals. Here, we show that AdhR, a σ 54 -dependent transcriptional activator, senses the intracellular redox status and controls alcohol synthesis in Clostridium beijerinckii AdhR provides a new example of a GAF domain coordinating a mononuclear non-heme iron to sense and transduce the redox signal. Our study reveals a previously unrecognized functional role of σ 54 in control of cellular redox balance and provides new insights into redox signaling and regulation in clostridia. Our results reveal AdhR as a novel engineering target for improving solvent production by C. beijerinckii and other solventogenic clostridia., (Copyright © 2020 American Society for Microbiology.)

Background: The σ54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ54 remain unknown. Results: For systematic analysis of the regulatory functions of σ54, we performed comparative genomic reconstruction of transcriptional regulons of σ54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ54 regulons between different Clostridiales species. It is proposed that σ54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species. Conclusions: This study reveals previously unrecognized functions of σ54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species. [ABSTRACT FROM AUTHOR]

Background: The σ 54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ 54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ 54 remain unknown., Results: For systematic analysis of the regulatory functions of σ 54 , we performed comparative genomic reconstruction of transcriptional regulons of σ 54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ 54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ 54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ 54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ 54 regulons between different Clostridiales species. It is proposed that σ 54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species., Conclusions: This study reveals previously unrecognized functions of σ 54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species.

Bacterial enhancer binding proteins (bEBPs) are transcription activators that belong to the AAA(+) protein family. They form higher-order self-assemblies to regulate transcription initiation at stress response and pathogenic promoters. The precise mechanism by which these ATPases utilize ATP binding and hydrolysis energy to remodel their substrates remains unclear. Here we employed mass spectrometry of intact complexes to investigate subunit dynamics and nucleotide occupancy of the AAA(+) domain of one well-studied bEBP in complex with its substrate, the σ(54) subunit of RNA polymerase. Our results demonstrate that the free AAA(+) domain undergoes significant changes in oligomeric states and nucleotide occupancy upon σ(54) binding. Such changes likely correlate with one transition state of ATP and are associated with an open spiral ring formation that is vital for asymmetric subunit function and interface communication. We confirmed that the asymmetric subunit functionality persists for open promoter complex formation using single-chain forms of bEBP lacking the full complement of intact ATP hydrolysis sites. Outcomes reconcile low- and high-resolution structures and yield a partial sequential ATP hydrolysis model for bEBPs., (© 2013. Published by Elsevier Ltd. All rights reserved.)

Bacterial enhancer binding proteins (bEBPs) are a subclass of the AAA(+) (ATPases Associated with various cellular Activities) protein family. They are responsible for σ(54)-dependent transcription activation during infection and function under many stressful growth conditions. The majority of bEBPs are regulated in their formation of ring-shaped hexameric self-assemblies via an amino-terminal domain through its phosphorylation or ligand binding. In contrast, the Escherichia coli phage shock protein F (PspF) is negatively regulated in trans by phage shock protein A (PspA). Up to six PspA subunits suppress PspF hexamer action. Here, we present biochemical evidence that PspA engages across the side of a PspF hexameric ring. We identify three key binding determinants located in a surface-exposed 'W56 loop' of PspF, which form a tightly packed hydrophobic cluster, the 'YLW' patch. We demonstrate the profound impact of the PspF W56 loop residues on ATP hydrolysis, the σ(54) binding loop 1, and the self-association interface. We infer from single-chain studies that for complete PspF inhibition to occur, more than three PspA subunits need to bind a PspF hexamer with at least two binding to adjacent PspF subunits. By structural modelling, we propose that PspA binds to PspF via its first two helical domains. After PspF binding-induced conformational changes, PspA may then share structural similarities with a bEBP regulatory domain., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)